1. Field of the Invention
This invention relates to the analysis or identification of biological samples using nucleic acid detection techniques, such as DNA amplification and sequencing. More particularly, the invention relates to ways of monitoring problems that can arise during such techniques, such as cross-contamination of materials from different sources, as well as enabling sample traceability and providing internal controls for such techniques.
2. Background Art
The technology of molecular biology has opened up number of applications for testing, analysis and diagnosis using nucleic acid techniques (see References 1, 2 and 3 of the References section below). This includes routine testing of clinical, veterinary, end plant samples, as well as development of drugs and specialty bio-pharmaceuticals.
In the early 1990s, a novel nucleic acid amplification technology, polymerase chain reaction (PCR), was made possible. Although the primary use of this technology was targeted at making millions of copies of DNA segments that could be cloned, PCR was later adopted in routine nucleic acid testing, e.g. testing for the presence of infectious microorganisms or specific genes or mutations. Presently, there are number of available assays that use conventional PCR as well as other modifications, such as reverse transcriptase PCR (RT-PCR, used to amplify sequences derived from RNA), ligase chain reaction (LCR), transcription mediated amplification (TMA), etc, to enable the amplification of target nucleic acid molecules having sequences of interest. In addition to target amplification technologies, there are number of signal amplification technologies, such as b-DNA and hybrid capture technology that are presently being used in routine testing. The use of nucleic acids techniques has made routine testing faster so that results can be obtained within hours rather than waiting days or weeks for bacterial cultures to grow.
Whatever the specific technology, routine testing warrants sensitivity (confidence that a negative is true negative) and specificity (confidence that a positive is a true positive). For example, nucleic acids based technologies have to conform to sensitivity and specificity requirements dictated by controlling authorities such as CLIA (Clinical laboratory Improvement Amendment, USA). In addressing these requirements, it has become routine to include, in each batch of testing, one sample without a target that would serve as negative control and one sample with the target as a true positive control, but this increases the complexity and cost of the testing operation. Furthermore, the controls may not be subjected to exactly the same conditions and environment as the actual samples.
Moreover, the fact that extremely small amounts of DNA or RNA may be amplified by PCR and similar technologies means that there is a real possibility of contamination of the samples (i.e. with amplified foreign DNA) employed for testing because small amounts of contaminants may also be amplified and yield an inaccurate result.
Also there is a need for traceability of samples because large numbers of samples are routinely tested and can easily be mixed up. Such sample mix-ups sometimes occur in hospitals and laboratories, so there is also a need for a way of confirming the origin or identity of a particular sample even if there is no contamination as such.
Additionally, ingredients in certain samples (e.g. urine) are known to inhibit target amplification processes, such as PCR or RNA polymerization (References 6 and 7), so there is a need to ensure that there are no inhibitory substances in the samples that could have that effect. In addressing the inhibitory substance issue in the past, artificial targets have been introduced into the samples where the artificial targets will have the same annealing site as that of the target, and the artificial targets produce an amplicon that carries an internal sequence that is different from that of the target (References 4 and 5). Using a third probe specific to this target will detect the artificial target ensuring there are no inhibitory substances. However, this procedure is quite comprised and may not always be reliable.
There is therefore a need for an improved means of monitoring nucleic acid testing techniques.